Antibody free ELISA-like assay for the detection of transcription factors based on double-stranded DNA thermostability.

Transcription factors (TFs) play critical roles in gene expression regulation and disease development. In this paper, we report an antibody free ELISA-like assay which could be used to analyze transcription factor NF-κB p50 with comparatively low cost and high throughput. This assay is based on the stabilization of a duplex DNA probe by binding with a transcription factor. The double-stranded DNA (dsDNA) probe immobilized on a 96-well plate was too short in length to stabilize its duplex structure at a relatively high temperature and would unwind into a single strand. In the presence of a target TF, the protein bound to a specific TF-binding site and prevented the specific dsDNA probe from being unzipped at the washing temperature, thus holding the chemiluminescence signal. This method could sensitively detect NF-κB p50 with a detection limit of 0.5 nM. The proposed strategy provides a convenient, cheap and high throughput detection method of TFs, which can potentially help the development of drug discovery and disease diagnosis.

Ultrasensitive Homogeneous Electrochemical Detection of Transcription Factor by Coupled Isothermal Cleavage Reaction and Cycling Amplification Based on Exonuclease III.

The assay and monitoring of transcription factors (TFs) has attracted extensive attention due to their important roles in regulation of gene expressions. Herein, a simple, low cost, rapid, and highly sensitive homogeneous electrochemical method utilizing the coupled isothermal cleavage reaction and cycling amplification based on exonuclease III (Exo III) was explored for the analysis of transcription factor NF-κB p50 in aqueous solution. In the assay, a 3'-methylene blue (MB)-labeled hairpin probe is designed, which can be opened up by the single stranded DNA (ssDNA) protected by NF-κB p50 from the Exo III cleavage, to trigger the subsequent Exo III-assisted digestion, thus a large amount of MB-labeled mononucleotides are liberated to result in the greatly amplified electrochemical signal. By virtue of this Exo III-assisted target recycling, the present assay allows the detection of NF-κB p50 at the picomolar level, which is an exciting level for TFs detection. Furthermore, this detection possesses excellent selectivity, demonstrating high application potential in biological system and convenient TFs' inhibitors screening. Comparing with the other reported strategies for TFs detection, this Exo III-assisted homogeneous electrochemical detection platform was just composed of one kind of enzyme and two DNA probes, offered a really simple and low-cost electrochemical detection for TFs assay.

NF-κB p50 activation associated with immune dysregulation confers poorer survival for diffuse large B-cell lymphoma patients with wild-type p53.

Dysregulated NF-κB signaling is critical for lymphomagenesis, however, the expression and clinical relevance of NF-κB subunit p50 in diffuse large B-cell lymphoma have not been evaluated. In this study, we analyzed the prognostic significance and gene expression signatures of p50 nuclear expression as a surrogate for p50 activation in 465 patients with de novo diffuse large B-cell lymphoma. We found that p50+ nuclear expression, observed in 34.6% of diffuse large B-cell lymphoma, predominantly composed of activated B-cell-like subtype, was an independent adverse prognostic factor in patients with activated B-cell-like diffuse large B-cell lymphoma. It was also an adverse prognostic factor in patients with wild-type TP53 independent of the activated B-cell-like and germinal center B-cell-like subtypes, even though p50 activation correlated with significantly lower levels of Myc, PI3K, phospho-AKT, and CXCR4 expression and less frequent BCL2 translocations. In contrast, in germinal center B-cell-like diffuse large B-cell lymphoma patients with TP53 mutations, p50+ nuclear expression correlated with significantly better clinical outcomes, and decreased p53, Bcl-2, and Myc expression. Gene expression profiling revealed multiple signaling pathways potentially upstream the p50 activation through either canonical or noncanonical NF-κB pathways, and suggested that immune suppression, including that by the immune checkpoint TIM-3 and that through leukocyte immunoglobulin-like receptors, but not antiapoptosis and proliferation, may underlie the observed poorer survival rates associated with p50+ nuclear expression in diffuse large B-cell lymphoma. In conclusion, these data show that p50 is important as a unique mechanism of R-CHOP-resistance in activated B-cell-like diffuse large B-cell lymphoma and in patients without TP53 mutations. The results also provide insights into the regulation and function of p50 in diffuse large B-cell lymphoma and its cross talk with the p53 pathway with important therapeutic implications.

Differential gene expression in Mycobacterium bovis challenged monocyte-derived macrophages of cattle.

A functional genomics approach was used to examine the immune response for transcriptional profiling of PBMC M. bovis infected cattle and healthy control cattle to stimulation with bovine tuberculin (purified protein derivative PPD-b). Total cellular RNA was extracted from non-challenged control and M. bovis challenged MDM for all animals at intervals of 6 h post-challenge, in response to in-vitro challenge with M. bovis (multiplicity of infection 2:1) and prepared for global gene expression analysis using the Agilent Bovine (V2) Gene Expression Microarray, 8 × 60 K. The pattern of expression of these genes in PPD bovine stimulated PBMC provides the first description of an M. bovis specific signature of infection that may provide insights into the molecular basis of the host response to infection. Analysis of these mapped reads showed 2450 genes (1291 up regulated and 1158 down regulated) 462 putative natural antisense transcripts (354 up-regulated and 108 down regulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). The results provided enrichment for genes involved top ten up regulated and down regulated panel of genes, including transcription factors proliferation of T and B lymphocytes. The highest differentially-expressed genes were associated to immune and inflammatory responses, immunity, differentiation, cell growth, apoptosis, cellular trafficking and regulation of lipolysis and thermogenesis. Microarray results were confirmed in infected cattle by RT qPCR to identify potential biomarkers TLR2, CD80, NFKB1, IL8, CXCL6 and ADORA3 of bovine tuberculosis.

Artifacts Arising from Using Leukocytic Fc Receptor Blocking Buffer.

We studied the effects of Human TruStain FcX buffer (Fcγ receptor blocking solution) in experiments on evaluation of TLR4 level with labeled monoclonal antibodies, intracellular immunofluorescent staining of NF-κB p50, and TNF-α synthesis on human isolated monocytes and whole blood cells. The influence of the blocking buffer on the measured parameters should be taken into account and appropriateness of its use in experiments on isolated cells and whole blood should be considered.

The NFKB1 polymorphism (rs4648068) is associated with the cell proliferation and motility in gastric cancer.

BACKGROUND: We have demonstrated previously that NFKB1 single nucleotide polymorphism (SNP) rs4648068 GG homozygote was associated with the increased risk of gastric cancer in Chinese Han population. In this study, we constructed the recombinant plasmid pGL3-AA, pGL3-GG, pGL3-AA-NFKB and pGL3-GG-NFKB to investigate the function of rs4648068 by cell biology experiments. METHODS: Quantitative real-time PCR was used to detect NFKB1 SNP rs4648068 genotype in the patients with gastric cancer. Anti-NF-κB1 p50 polyclonal antibodies were used for immunohistochemical analysis of the tissue specimens. The subsection of NFKB1 containing the promoter site and adjacent three consecutive exons were obtained by PCR technique and subcloned into the vector pGL3-Basic. Dual-Luciferase reporter assay was used to detect the transcriptional activity of the constructed promoter. Effects of transcription factor NFKB1 on C/EBPß expression were determined by chromatin immunoprecipitation and Western analysis. Furthermore, proliferation and invasion ability of the transduced cell were also measured and compared. RESULTS: Intensive staining for p50 expression was observed in the tissues of GG genotype patients, compared with those of GA group and AA genotype patients. The transcriptional activity of rs4648068 (A > G) by dual-Luciferase reporter assay suggested that the luciferase activity of homozygote group (pGL3-GG) was greater than that of the control (pGL3-AA), especially at the stimulation of LPS. We found that the luciferase activity was also influenced by pGL3-GG levels. The effects of NFKB1 rs4648068 were enhanced by rs4648065 on the transduced cells. The interaction between NFKB1 promoter nucleotide sequence and C/EBPß was regulated by the functional SNP rs4648068 in SGC-7901 cells. Our data indicated that the transduction of pGL3 expression plasmid pGL3-GG-NFKB improved the proliferation and motility of gastric cancer cells. Correspondingly, the homozygote GG of SNP rs4648068 strengthened the transcriptional activity of NFKB1 and influenced the cell biological activity. CONCLUSION: The transcriptional activity of NFKB1 was associated with SNP rs4648068, and this functional SNP site has the important effects on cell proliferation and motility.

Steroid treatment can inhibit nuclear localization of members of the NF-κB pathway in human disc cells stimulated with TNF-α.

Steroid applications are able to repress inflammatory activity in various conditions, including herniation of the nucleus pulposus (HNP), by inhibiting tumour necrosis factor (TNF)-α, but the effects of long-term use are unknown. Here, we investigated the effect of dexamethasone (DEXA) on TNF-α-stimulated intervertebral disc cells by monitoring the expression and localization of NF-κB in the cytoplasm and nucleus. Cultured human intervertebral disc cells were left untreated or treated with only TNF-α, only DEXA, or with TNF-α and DEXA simultaneously. Cytoplasmic and nuclear proteins were extracted and Western blotted after 10 min, 1 or 2 h, to evaluate the expression of p50, p65, p52, and p100 (components of NF-κB). Immunofluorescence analysis was used to determine the subcellular localization of the proteins at 1 h. DEXA had limited effects on NF-κB expression in TNF-α-stimulated disc cells within the first 10 min. At 1 h, DEXA prevented the TNF-α-stimulated translocation of p50, p52, and p65. After 2 h, DEXA reduced the nuclear expression of p50, p65, and p52. Thus, DEXA resulted in delayed expression of NF-κB components and inhibited the translocation of p50, p52, and p65 to the nucleus, which would prevent expression of the corresponding genes. Therefore, following stimulation with TNF-α, transcriptional regulation of NF-κB in disc cells is mainly mediated via the classical pathway, but also to some extent via the alternative pathway. Hence, blockade of sub-acute inflammatory changes in HNP can be achieved by early injection of steroids, whereas long-term injection of a steroid may initiate NF-κB autophosphorylation.

Low expression of glucocorticoid receptor a in oral lichen planus correlates with activation of nuclear factor κB: a preliminary study.

BACKGROUND: Emerging evidence indicates that the interaction between glucocorticoid receptor α (GRα) and nuclear factor κB (NF-κB) is a key pathogenetic cross talk in the autoimmune and inflammatory disorders. The objective of this study was to determine the GRα expression in patients with oral lichen planus (OLP) and investigate its correlation with NF-κB in OLP. METHODS: We compared the expression of GRα and NF-κB in oral biopsy specimens from patients with OLP(n = 32) against normal controls (n = 12) and investigated the correlation between the expression of GRα and NF-κB in OLP. RESULTS: Immunohistochemistry showed that GRα mainly expressed in the cytoplasm of keratinocytes of basal and spinosum layer of OLP. Both real-time quantitative PCR and Western blots revealed that the mRNA and protein expression levels of GRα were decreased compared with normal controls (both P < 0.001). Conversely, those levels of nuclear factor-kappa B (NF-κB) were increased compared with normal controls (both P < 0.001). Importantly, a significant inverse correlation between the GRα and NF-κB was found (P < 0.05). CONCLUSIONS: Our findings demonstrated that low expression of GRα in OLP correlates with activation of NF-κB, which indicates that the cross talk between GRα and NF-κB in OLP may become a new therapeutic target and represent a new approach to explore the pathogenesis of OLP.

Real-time detection of transcription factors using target-converted helicase-dependent amplification assay with zero-background signal.

Highly sensitive detection of transcription factors is essential to the evaluation of cellular development and the disease state. However, so far most detection methods are usually laborious and time-consuming with a poor sensitivity. Here, we demonstrate a simple and ultrasensitive approach for transcription factor detection based on the target-converted helicase-dependent amplification assay. We employ a hairpin probe bearing the transcription factor binding site to convert the protein signal to the DNA signal, which can be further amplified by helicase-dependent amplification. With the digestion of excess probes by the exonucleases and the subsequent one primer-triggered high fidelity amplification, zero-background signal can be achieved in the absence of a transcription factor. This method exhibits excellent specificity and high sensitivity with the detection limit of 9.3× 10(-13) M and the detection range over 4 orders of magnitude, which is superior to most currently used approaches for transcription factor detection. Moreover, the proposed method has significant advantages of simple, rapid, and low cost without the need of any labeled DNA probes and might be extended to selectively detect various DNA-binding proteins by simply changing the binding-site sequences of hairpin probes.

Glycated matrix up-regulates inflammatory signaling similarly to Porphyromonas gingivalis lipopolysaccharide.

BACKGROUND AND OBJECTIVE: Hyperglycemia and advanced glycation end-products (AGEs) have been hypothesized as the etiologic factors of diabetic periodontitis. The aim of this study was to clarify in greater detail the patterns of AGE-mediated periodontal inflammation under various physiological conditions. MATERIAL AND METHODS: The deposition of AGEs and expression of the receptor for AGEs (RAGE) were identified by immunohistochemistry in Sprague-Dawley rats with experimentally induced periodontitis or diabetes. Human periodontal ligament cells (PDLCs) and mesenchymal stem cells (MSCs) were cultured under simulated conditions of hyperglycemia, Porphyromonas gingivalis lipopolysaccharide (LPS) stimulation and matrix glycation. Cell viability and expression of toll-like receptors (TLRs), Rage, an inflammatory signaling initiator (nuclear factor kappa light chain enhancer of activator ß cells), an oxidative stressor (heme oxygenase-1) and collagen synthesis (type I and type IV) genes were evaluated. RESULTS: The deposition of AGEs and the expression of Rage were evident in the inflamed periodontal tissues in all rats and appeared to be enhanced in rats with diabetes. Matrix glycation augmented cytotoxicity, up-regulated RAGE and TLRs in both PDLCs and MSCs, and significantly activated downstream inflammatory signaling in MSCs. Oxidative stress was significantly increased under matrix glycation in both PDLCs and MSCs and was significantly increased at a high-glucose concentration in MSCs. A consistent decrease in expression of type I and type IV collagens was observed in MSCs, but a delayed reduction was noted in PDLCs. CONCLUSIONS: Matrix glycation modulated cell behavior to induce inflammation equivalent to that produced by incubation with P. gingivalis LPS. Periodontal inflammation also led to matrix glycation, thus demonstrating a definite interaction between diabetes and periodontitis.

A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB.

Transcription factors are involved in a number of important cellular processes. The transcription factor NF-κB has been linked with a number of cancers, autoimmune and inflammatory diseases. As a result, monitoring transcription factors potentially represents a means for the early detection and prevention of diseases. Most methods for transcription factor detection tend to be tedious and laborious and involve complicated sample preparation, and are not practical for routine detection. We describe herein the first label-free luminescence switch-on detection method for transcription factor activity using Exonuclease III and a luminescent ruthenium complex, [Ru(phen)(2)(dppz)](2+). As a proof of concept for this novel assay, we have designed a double-stranded DNA sequence bearing two NF-κB binding sites. The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit. In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed. The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity. This will allow the rapid and low cost identification and development of novel scaffolds for the treatment of diseases caused by the deregulation of transcription factor activity.

Profile of NF-κBp(65/NFκBp50) among prostate specific antigen sera levels in prostatic pathologies.

AIM: The aim of this work was to characterise the immunoexpression of NF-κB (p50/p65) in human prostatic pathologies and to study its profiles of activation among sera prostate specific antigen antigen (PSA) according the three groups: 0-4ng/mL, 4-20ng/mL and >20ng/mL. PATIENTS AND METHODS: Twenty-four men with benign prostate hyperplasia (BPH); 19 men with prostate cancer (PC) and five men with normal prostates (NP). Immunohistochemical and western blot analysis was performed. Serum levels of PSA were assayed by immulite autoanalyser. RESULTS: In BPH and PC samples, immunoexpressions were observed for NF-κBp65 and NF-κBp50; while in NP samples, only were detected NF-κBp50. PC samples showed immunoreactions to NF-κBp65 and NF-κBp50 more intense (respectively 24.18±0.67 and 28.23±2.01) than that observed in BPH samples (respectively18.46±2.04 and 18.66±1.59) with special localisation in the nucleus. Different profiles of NF-κBp65 immunoexpressions were observed and BPH patients with sera PSA levels between 0-4ng/mL presented a significant weak percentage compared to BPH patients with sera PSA levels between 4-20ng/mL and >20ng/mL. No immunoreactions to NF-κBp65 were observed in PC patients with sera PSA levels between 4-20ng/mL. CONCLUSION: The sensibility of both NF-κB and PSA to inflammation allowed confirming the relationship between these two molecules and its involvement in prostatic diseases progression (inflammatory and neoplasic).

Hairpin-like fluorescent probe for imaging of NF-κB transcription factor activity.

Three oligodeoxyribonucleotides (ODN) covalently labeled with near-infrared (NIR) fluorochromes were synthesized and characterized with a goal of comparing in vitro a hairpin-based and a duplex-based FRET probe designed for the detection of human recombinant NF-κB p50/p65 heterodimer binding to DNA. Using deoxyguanosine phosphoramidite with a phosphorus-linked aminoethylene (diethylene glycol) hydrophilic linker, we synthesized ODNs with internucleoside reactive sites. The hairpin loop amino linker was modified with IRDye 800CW (FRET acceptor), and the 3'-end was modified with Cy5.5 (FRET donor) using a dithio-linker. To obtain a duplex probe, we conjugated Cy5.5 and 800CW to complementary strands at the distance of ten base pairs in the resultant duplex. No quenching of dyes was observed in either probe. The FRET efficiency was higher in the duplex (71%) than in the hairpin (56%) due to a more favorable distance between the donor and the acceptor. However, the hairpin design allowed more precise ratiometric measurement of fluorescence intensity changes as a result of NF-κB p50/p65 binding to the probe. We determined that as a result of binding there was a statistically significant increase of fluorescence intensity of Cy5.5 (donor) due to a decrease of FRET if normalized by 800CW intensity measured independently of FRET. We conclude that the hairpin based probe design allows for the synthesis of a dual fluorescence imaging probe that renders signal changes that are simple to interpret and stoichiometrically correct for detecting transcription factor-DNA interactions.

Annexin A4 interacts with the NF-kappaB p50 subunit and modulates NF-kappaB transcriptional activity in a Ca2+-dependent manner.

Previously, we identified annexin A4 (ANXA4) as a candidate substrate of caspase-3. Proteomic studies were performed to identify interacting proteins with a view to determining the roles of ANXA4. ANXA4 was found to interact with the p105. Subsequent studies revealed that ANXA4 interacts with NF-kappaB through the Rel homology domain of p50. Furthermore, the interaction is markedly increased by elevated Ca(2+) levels. NF-kappaB transcriptional activity assays demonstrated that ANXA4 suppresses NF-kappaB transcriptional activity in the resting state. Following treatment with TNF-alpha or PMA, ANXA4 also suppressed NF-kappaB transcriptional activity, which was upregulated significantly early after etoposide treatment. This difference may be due to the intracellular Ca(2+) level. Additionally, ANXA4 translocates to the nucleus together with p50, and imparts greater resistance to apoptotic stimulation by etoposide. Our results collectively indicate that ANXA4 differentially modulates the NF-kappaB signaling pathway, depending on its interactions with p50 and the intracellular Ca(2+) ion level.

Electrochemiluminescence platform for transcription factor diagnosis by using CRISPR-Cas12a trans-cleavage activity.

Herein, we exploited the double-stranded DNA (dsDNA) binding property of transcription factor (TF), combined with the trans cleavage characteristic of CRISPR-Cas12a, for the detection of NF-κB p50.

Entropy-driven electrochemiluminescence ultra-sensitive detection strategy of NF-κB p50 as the regulator of cytokine storm.

2019 novel coronavirus (2019-nCoV) with strong contagion in the crowd, has ravaged worldwide and severely impacts the human health and epidemic prevention system, by producing a series of significant stress reactions in the body to induce further cytokine storm. Transcription factors (TFs) served as essential DNA binding proteins play an integral role in regulating cytokine storm, and the detection of it in the human coronavirus environment provides especially valuable approaches to diagnosis and treatment of 2019-nCoV and development of antiviral drugs. In this work, an entropy-driven electrochemiluminescence (ECL) biosensor was constructed for ultra-sensitive bioassay of NF-κB p50. The strategy primarily capitalizing the splendid double-stranded DNA (dsDNA) binding properties of transcription factors, employing GOAu-Ru composite material as ECL emitter, utilizing entropy-driven reactions for signal amplification method, offered a repeatable proposal for TFs detection. In the absence of TFs, the released DNA1 further went in the entropy-driven reaction, contributing to an "ECL off" state. However, in the presence of TFs, the dsDNA avoided being digested, which blocked DNA1 for participating in the entropy-driven reaction, and the system exhibited an "ECL on" state. Most importantly, the ECL bioanalytical method denoted broad application prospects for NF-κB p50 detection with a lower detection limit (9.1 pM).

Increased IkappaB alpha expression is essential for the tolerogenic property of TGF-beta-exposed APCs.

IkappaB alpha is an inhibitor of the transcriptional factor NF-kappaB, and it is an essential component of the signaling pathways that lead to expression of inflammatory molecules. These include cytokines and costimulatory molecules associated with antigen presentation in an inflammatory immune response. In this study, we report that antigen-presenting cells exposed to TGF-beta induce peripheral tolerance by increasing IkappaB alpha expression. Exposure of antigen presenting cells (APCs) to TGF-beta is known to impair their ability to secrete IL-12, and such impairment correlated with reduced NF-kappaB activity as indicated by significantly reduced nuclear levels of p50, an essential subunit of NF-kappaB for IL-12 transcription. Blockade of increased nuclear IkappaB alpha in APCs by expression of small interfering RNA molecules (siRNAs) targeting IkappaB alpha transcripts prevented IL-12 impairment and the decline in nuclear p50 levels. Furthermore, such IkappaB alpha blockade also interfered with the tolerogenic property of TGF-beta-exposed APCs. However, increased expression of IkappaB alpha in APCs, independent of TGF-beta exposure, reduced nuclear p50 levels and permitted tolerance induction by APCs. Thus, our findings attribute a direct and significant role to IkappaB alpha in the tolerogenic potential of APCs. Increased IkappaB alpha expression in APCs may therefore offer a therapeutic approach to achieve antigen-specific immunomodulation.

Cytotoxic and NF-kappaB inhibitory constituents of Artocarpus rigida.

Four new prenylated flavonoids (1-4), a new stilbenoid (5), and nine known compounds were isolated from the twigs of Artocarpus rigida, collected in Indonesia. The structures of the new compounds were determined by analysis of their spectroscopic data, and the absolute configuration at C-12 of 1 and 2 and the known compounds artonin O (6), artobiloxanthone (7), and cycloartobiloxanthone (8) was determined from their CD and NMR spectroscopic data. Several of the compounds obtained were cytotoxic toward HT-29 human colon cancer cells, with the most potent being compound 2 and the known compounds 6 and 8. Of the substances obtained, compounds 1 and 7 were the most active in the NF-kappaB p50 and p65 assay, respectively.

Development of a bidirectional isothermal amplification strategy for the sensitive detection of transcription factors in cancer cells.

We developed a new strategy to sensitively detect transcription factors (TFs) based on the integration of a bidirectional isothermal exponential amplification reaction (EXPAR) with endonuclease IV (endo IV)-assisted cycle digestion of signal probes. This assay exhibits ultrahigh sensitivity with a detection limit of 1.29 × 10-14 M, and it can measure endogenous NF-κB p50 in HeLa cell extracts. Moreover, this strategy can be applied to screen TF inhibitors and detect other TFs by simply changing the TF-binding sequence.

Dual-Wavelength Electrochemiluminescence Ratiometric Biosensor for NF-κB p50 Detection with Dimethylthiodiaminoterephthalate Fluorophore and Self-Assembled DNA Tetrahedron Nanostructures Probe.

In this work, we fabricated a dual-wavelength electrochemiluminescence ratiometric biosensor based on electrochemiluminescent resonance energy transfer (ECL-RET). In this biosensor, Au nanoparticle-loaded graphitic phase carbon nitride (Au-g-C3N4) as a donor and Au-modified dimethylthiodiaminoterephthalate (TAT) analogue (Au@TAT) as an acceptor were investigated for the first time. Besides, tetrahedron DNA probe was immobilized onto Au-g-C3N4 to improve the binding efficiency of the transcription factor and ECL ratiometric changes on the basis of the ratio of ECL intensities at 595 and 460 nm, which were obtained through the formation of a sandwich structure of DNA probe-antigen-antibody. Our biosensor achieved the assay of NF-κB p50 with a detection limit of 5.8 pM as well as high stability and specificity.